Volume 34, Issue 6, Pages 652-662 (June 2009) KSR2 Is a Calcineurin Substrate that Promotes ERK Cascade Activation in Response to Calcium Signals  Michele K. Dougherty, Daniel A. Ritt, Ming Zhou, Suzanne I. Specht, Daniel M. Monson, Timothy D. Veenstra, Deborah K. Morrison  Molecular Cell  Volume 34, Issue 6, Pages 652-662 (June 2009) DOI: 10.1016/j.molcel.2009.06.001 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 KSR2 Functions as a MAPK Scaffold for the ERK Cascade (A) Schematic depiction of KSR1 and KSR2. Amino acids and motifs involved in binding interactions are indicated as are the five conserved KSR domains (CA1–5). (B) KSR complexes isolated from cycling 293T cells were resolved by SDS-PAGE and stained with Coomassie brilliant blue. The control samples were prepared from untransfected cell lysates. Various proteins identified in the KSR complexes are indicated. (C) Pyo-KSR2 complexes were isolated from serum-starved or PDGF-treated NIH 3T3 cells and examined for the presence of B-Raf, MEK, ERK, active pMEK, and active pERK by immunoblot analysis. (D) Stage VI Xenopus oocytes were injected with the indicated amount of mRNA encoding KSR2. Oocytes were later injected with 3 ng of RasV12 mRNA and scored for maturation (as determined by germinal vesicle breakdown; GVBD) 5 and 8 hr following Ras injection. Oocyte lysates were prepared at the 8 hr time point and examined by immunoblot analysis as indicated. Molecular Cell 2009 34, 652-662DOI: (10.1016/j.molcel.2009.06.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Calcineurin Selectively Interacts with KSR2 (A) Pyo-tagged full-length (FL) and truncated N- and C-terminal KSR1 and KSR2 proteins were isolated from cycling COS-7 cells and examined for the presence of PP2Ac, CN-A, and CN-B. (B) Endogenous KSR2 complexes isolated from brain tissue of WT or KSR1−/− mice were examined for the presence of CN-B (upper). Serum-starved COS-7 cells expressing Pyo-KSR2 were treated with 100 ng/ml EGF (5 min), 1 μM ionomycin (15 min)/50 ng/ml PMA (5 min), 1 μM ionomycin (15 min), or 5 μM CsA (15 min). KSR2 complexes were isolated and examined for the presence of CN-B (lower). (C and D) Various Pyo-KSR2 mutant complexes were isolated from cycling COS-7 cells and examined for the presence of CN-B or PP2Ac. Sequence alignment of the LxVP motif found in NFAT proteins and KSR2. (E) GST-tagged KSR2 303-413 or KSR2 303-413-LxVPm proteins were examined for their ability to bind CN-B. Calcineurin was incubated with the indicated peptides, and activity assays were performed using phosphorylated RII as a substrate. KSR2 peptides were cleaved from GST, and AID is a peptide derived from the autoinhibitory domain of calcineurin. Results represent the range of two independent experiments performed in triplicate. Molecular Cell 2009 34, 652-662DOI: (10.1016/j.molcel.2009.06.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Ca2+-Signaling Modulates the Scaffolding Activity and Localization of KSR2 (A) Serum-starved COS-7 cells expressing Pyo-tagged WT-KSR2, LxVPm-KSR2, or KSR1 were treated as indicated. KSR complexes and total cell lysates were examined for the presence of active pERK. (B and C) Serum-starved COS-7 cells expressing WT-KSR2, LxVPm-KSR2, or KSR1 were treated as indicated prior to fixation. Localization of the KSR proteins and active pERK was determined by immunofluorescent staining. Molecular Cell 2009 34, 652-662DOI: (10.1016/j.molcel.2009.06.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 KSR2 Is a Calcineurin Substrate (A) Quiescent COS-7 expressing Pyo-tagged WT-KSR2, LxVPm-KSR2, or KSR1 were metabolically labeled with [32P]orthophosphate. Untreated or CsA-treated cells were stimulated with PMA/ionomycin prior to lysis. Immunoprecipitated KSR proteins were digested with trypsin and the tryptic phosphopeptides were separated by HPLC. Shown are the profiles of radioactivity collected in the HPLC fractions. Phosphorylated residues are indicated. (B) Localization of Pyo-KSR2 proteins in cycling COS-7 cells was determined by immunofluorescent staining. Pyo-KSR2 mutant complexes were isolated from cycling COS-7 cells and examined for the presence of 14-3-3. Molecular Cell 2009 34, 652-662DOI: (10.1016/j.molcel.2009.06.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Endogenous KSR2 Is Regulated by Ca2+ Signals in INS1 and NG108 Cells (A) Lysates prepared from various mouse tissues and cell lines were examined for endogenous KSR2 expression. An asterisk (∗) indicates a nonspecific band. (B) Untreated or CsA-treated NG108 and INS1 cells were stimulated with either glucose (Glc) or KCl for 20 min. Endogenous KSR2 complexes were isolated and examined for the presence of active pERK. (C) Localization of endogenous KSR2 in INS1 and NG108 cells was determined by immunofluorescent staining. (D) INS1 and NG108 cells were metabolically labeled with [32P]orthophosphate and treated as indicated. Endogenous KSR2 proteins were isolated and examined by HPLC analysis. Molecular Cell 2009 34, 652-662DOI: (10.1016/j.molcel.2009.06.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Depletion of KSR2 Impairs Ca2+-Mediated ERK Signaling in NG108 and INS1 Cells (A) NG108 cells transfected with control, KSR1-, or KSR2-siRNAs were treated as indicated and then examined for KSR and pERK levels. Lentivirus-infected cells expressing either the pLKO vector or pLKO-KSR2 shRNA were also examined. Tubulin levels are shown as a protein loading control. (B) Control and KSR1- or KSR2-depleted INS1 cells were examined as in (A). Cells were also were examined for insulin secretion following culture for 2 hr in media containing either 2.5 or 15 mM glucose. Results represent the range of three independent experiments performed in duplicate. Molecular Cell 2009 34, 652-662DOI: (10.1016/j.molcel.2009.06.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Calcineurin Binding Is Required for Full KSR2 Function in KCl-Treated NG108 Cells NG108 cells expressing pLKO-KSR2 shRNA were transfected with plasmids encoding either GFP or resistant Pyo-tagged WT- or LxVPm KSR2. Cells were then examined for pERK and KSR2 levels following treatment with 60 mM KCl for 20 min (A) or for neurite formation following growth for 3 days in media containing 60 mM KCl (B). As a control, NG108 cells expressing the pLKO vector were also examined. Molecular Cell 2009 34, 652-662DOI: (10.1016/j.molcel.2009.06.001) Copyright © 2009 Elsevier Inc. Terms and Conditions